Correction: Embryos of Robertsonian Translocation Carriers Exhibit a Mitotic Interchromosomal Effect That Enhances Genetic Instability during Early Development.

[This corrects the article DOI: 10.1371/journal.pgen.1003025.].

Sperm Mitochondrial DNA Copy Number Is Not a Predictor of Intracytoplasmic Sperm Injection (ICSI) Cycle Outcomes.

This study is to determine if sperm mitochondrial DNA copy number (mtDNA CN) is associated with fertilization, blastulation, blastocyst euploidy, and live birth rates in in vitro fertilization (IVF) with ICSI cycles. This is a cohort study conducted on stored sperm samples which were collected prospectively and used to create blastocysts transferred in a couple's first ICSI transfer cycle between 2007 and 2013 at a single large infertility center. Samples from ICSI cycles utilizing surgical or cryopreserved sperm or day 3 embryo biopsy were excluded. The primary outcome was live birth rate. Secondary outcomes included fertilization, usable blastocyst development, and blastocyst euploidy rates. Unique sperm samples used to create transferred embryos were identified. Mitochondrial DNA CN was evaluated using TaqMan® quantitative real-time polymerase chain reaction (qPCR) assays normalized to a nuclear control for relative quantitation. Linear regression and mixed effects logistic regression used were appropriate. A total of 2062 unique sperm samples used to create transferred embryos were included. Lower relative sperm mtDNA content was associated with increased pre-wash sperm motility (p < 0.001). No significant association was identified between sperm mtDNA CN and fertilization (p = 0.40), usable blastocyst development (p = 0.36), blastocyst euploid (p = 0.10), and live birth rates (p = 0.42) while adjusting for sperm pre-wash motility and maternal age. Sperm mtDNA CN is not prognostic of fertilization, usable blastocyst development, euploidy and live birth rates in an infertile population undergoing IVF with ICSI.

Clinical outcomes after the transfer of blastocysts characterized as mosaic by high resolution Next Generation Sequencing- further insights.

OBJECTIVE: To determine the pregnancy outcome potential of euploid, mosaic and aneuploid embryos. DESIGN: Retrospective study. SETTING: Reference genetics laboratories. PATIENT(S): 2654 PGT-A cycles with euploid characterized embryo transfers, 253 PGT-A cycles with transfer of embryos characterized as mosaic, and 10 PGT-A cycles with fully abnormal embryo transfers. INTERVENTION(S): Blastocysts were assessed by trophectoderm (TE) biopsy followed by PGT-A via array CGH or NGS. MAIN OUTCOME MEASURE(S): Implantation, miscarriage, ongoing implantation rates (OIR), and karyotype if available, were compared between different embryo groups, and between the two PGT-A techniques. RESULTS: The Ongoing Pregnancy Rate (OPR)/transfer was significantly higher for NGS-classified euploid embryos (85%) than for aCGH ones (71%) (p < 0.001), but the OPR/cycle was similar (63% vs 59%). NGS-classified mosaic embryos resulted in 37% OPR/cycle (p < 0.001 compared to euploid). Mosaic aneuploid embryos with <40% abnormal cells in the TE sample had an OIR of 50% compared to 27% for mosaics with 40-80% abnormal cells in the TE, and 9% for complex mosaic embryos. All the karyotyped ongoing pregnancies (n = 29) were euploid. Transfers of embryos classified as aneuploid via aCGH (n = 10) led to one chromosomally abnormal pregnancy. CONCLUSION(S): NGS-classified euploid embryos yielded higher OIRs but similar OPRs/cycle compared to aCGH. NGS-classified mosaic embryos had reduced potential to reach term, compared to euploid embryos. If they did reach term, those with karyotype results available were euploid. Embryos carrying uniform aneuploidies affecting entire chromosomes were mostly unable to implant after transfer, and the one that implanted ended up in a chromosomally abnormal live birth.

The cytogenetic constitution of human blastocysts: insights from comprehensive chromosome screening strategies.

BACKGROUND: Embryos that are able to form blastocysts have succeeded in activating their genome and differentiating into two cell types-an external layer of trophectoderm cells, which will go on to form extra-embryonic tissues such as the placenta, and the inner cell mass, which will give rise to the embryo proper. Culturing embryos to the blastocyst stage has become an increasingly popular IVF practice over the past decade. Additionally, it has been proposed that the identification and transfer of euploid blastocysts could significantly improve IVF outcomes. Toward this end, comprehensive molecular cytogenetic methods have been developed. The application of such methods in both clinical and research contexts has yielded cytogenetic data from large numbers of blastocysts. Questions have been raised concerning the implantation potential of blastocysts diagnosed 'euploid' or 'aneuploid', and there has been particular debate over the detection and viability of embryos categorized as 'mosaic'-composed of a mixture of normal and aneuploid cells. OBJECTIVE AND RATIONALE: This review aims to summarize data from studies using comprehensive molecular cytogenetic methods to examine blastocyst-stage embryos, describing current knowledge related to rates of euploidy, uniform aneuploidy and mosaicism. Issues associated with the developmental capacity of blastocysts of different cytogenetic constitutions will also be addressed. Guidelines on the clinical management of blastocysts with varying chromosome complements will be considered. SEARCH METHODS: Rates of euploidy, uniform aneuploidy (in which all cells have the same abnormal karyotype) and mosaicism were determined via a thorough literature search (PubMed). The keywords used in the search were as follows: preimplantation embryo development, blastocyst stage, embryonic aneuploidy, meiotic chromosome malsegregation, post-zygotic chromosome malsegregation, comprehensive chromosome screening, array comparative genomic hybridization, single-nucleotide polymorphism array, next-generation sequencing, embryo mosaicism and implantation of mosaic embryos. Relevant articles written in English and published up to March 2018 were reviewed. OUTCOMES: Different types of aneuploidy, including some complex forms, are capable of persisting to the blastocyst stage. As expected, euploidy rates decreased with advancing female age, whereas uniform aneuploidy increased. Analysis of multiple cells from individual blastocysts revealed that most of those classified 'abnormal' contained no euploid cells (due to meiotic errors arising in the gametes and therefore present in every cell), some having additional mosaic (post-fertilization, mitotic) errors. Blastocysts with a mix of normal and aneuploid cells were observed less frequently than other classes of embryo. The transfer of embryos with diploid-aneuploid mosaic biopsy specimens is reportedly associated with higher miscarriage and lower implantation rates, compared to embryos in which only euploid cells are detected. WIDER IMPLICATIONS: Detailed investigations into the chromosome constitution of human blastocysts suggest that a significant proportion is euploid in every cell, although the exact fraction is strongly influenced by female age. These findings do not support the notion that mosaic chromosome abnormalities are a natural part of embryo development. Mosaic aneuploidies, arising post-zygotically, were detected by various different comprehensive molecular cytogenetic methods, suggesting that the majority of these represent genuine findings. However, it remains possible that certain comprehensive molecular cytogenetic methods may carry a risk of mosaicism being incorrectly assigned, in a minority of samples, as a result of technical artifact. This may be a consequence of degraded DNA in the trophectoderm biopsy or other technical issues. According to published studies, blastocysts considered to have uniform aneuploidy and, to a lesser extent, those with mosaic abnormalities were associated with poorer clinical outcomes in comparison with euploid embryos.

Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.

STUDY QUESTION: Can quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes? SUMMARY ANSWER: This study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential. WHAT IS KNOWN ALREADY: Better methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability-higher levels of mtDNA associated with reduced implantation potential. STUDY DESIGN, SIZE, DURATION: mtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner-i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation. MAIN RESULTS AND THE ROLE OF CHANCE: mtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P < 0.0001). LIMITATIONS REASONS FOR CAUTION: To determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion. WIDER IMPLICATIONS OF THE FINDINGS: This is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by institutional funding (Reprogenetics UK and Reprogenetics). DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. None of the authors have any competing interests.

Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing.

OBJECTIVE: To determine the pregnancy outcome potential of mosaic embryos, detected by means of preimplantation genetic screening (PGS) with the use of next-generation sequencing (NGS). DESIGN: Retrospective study. SETTING: Genetics laboratories. PATIENT(S): PGS cycles during which either mosaic or euploid embryos were replaced. INTERVENTION(S): Blastocysts were biopsied and processed with the use of NGS, followed by frozen embryo transfer. Trophectoderm (TE) biopsies were classified as mosaic if they had 20%-80% abnormal cells. MAIN OUTCOME MEASURE(S): Implantation, miscarriage rates, and ongoing implantation rates (OIRs) were compared between euploid and types of mosaic blastocysts. RESULT(S): Complex mosaic embryos had a significantly lower OIR (10%) than aneuploidy mosaic (50%), double aneuploidy mosaic (45%), and segmental mosaic (41%). There was a tendency for mosaics with 40%-80% abnormal cells to have a lower OIR than those with <40% (22% vs. 56%). However, few embryos (n = 34) with a mosaic error in 40%-80% of the TE sample were replaced. There was no difference between monosomic and trisomic mosaics or between entire chromosome mosaicism or segmental mosaicism. Implantation rates were significantly higher (70% vs. 53%), miscarriage rates lower (10% vs. 25%), and OIRs higher (63% vs. 40%) after euploid embryo transfer than after mosaic embryo transfer. CONCLUSION(S): Forty-one percent of mosaic embryos produced an ongoing implantation. Complex mosaic blastocysts had a lower OIR than other mosaics. Mosaic monosomies performed as well as mosaic trisomies and mosaic segmental aneuploidies. The results suggest that embryos with >40% abnormal cells and those with multiple mosaic abnormalities (chaotic mosaics) are likely to have lower OIRs and should be given low transfer priority.

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts.

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling.

Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.

Polymorphisms in the MTHFR gene influence embryo viability and the incidence of aneuploidy.

MTHFR is an important enzyme in the metabolism of folic acid and is crucial for reproductive function. Variation in the sequence of MTHFR has been implicated in subfertility, but definitive data are lacking. In the present study, a detailed analysis of two common MTHFR polymorphisms (c.677C>T and c.1298A>C) was performed. Additionally, for the first time, the frequencies of different MTHFR alleles were assessed in preimplantation embryos. Several striking discoveries were made. Firstly, results demonstrated that maternal MTHFR c.1298A>C genotype strongly influences the likelihood of a pregnancy occurring, with the 1298C allele being significantly overrepresented amongst women who have undergone several unsuccessful assisted reproductive treatments. Secondly, parental MTHFR genotypes were shown to affect the production of aneuploid embryos, indicating that MTHFR is one of the few known human genes with the capacity to modulate rates of chromosome abnormality. Thirdly, an unusual deviation from Hardy-Weinberg equilibrium was noted for the c.677C>T polymorphism in subfertile patients, especially those who had experienced recurrent failure of embryo implantation or miscarriage, potentially explained by a rare case of heterozygote disadvantage. Finally, a dramatic impact of the MTHFR 677T allele on the capacity of chromosomally normal embryos to implant is described. Not only do these findings raise a series of interesting biological questions, but they also argue that testing of MTHFR could be of great clinical value, identifying patients at high risk of implantation failure and revealing the most viable embryos during in vitro fertilisation (IVF) cycles.

Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability.

Mitochondria are the key regulators of multiple vital cellular processes, including apoptosis, calcium homeostasis, and the generation of ATP via the metabolic pathway known as oxidative phosphorylation. Unlike other cellular organelles, mitochondria contain one or more copies of their own genome (mtDNA). The mtDNA encodes a total of 13 genes with critical functions in cellular metabolism. The energy required to support the normal progress of preimplantation embryo development is provided in the form of ATP generated by the mitochondria. It has been suggested that cellular bioenergetic capacity and suboptimal levels of mitochondria-generated ATP could contribute to a variety of embryo developmental defects, and therefore adversely affect in vitro fertilization success rates. During this review, we discuss the role of mitochondria and their genome during oogenesis and early embryo development. We also assess whether analysis of mitochondria and their genome could be used as biomarkers to determine oocyte quality and embryo viability.